Pesticide residue detection test strip as well as preparation method and application thereof

Abstract

The invention relates to a pesticide residue detection test strip as well as a preparation method and application thereof, in particular to a rapid detection technology based on an enzyme inhibition method and a chitosan non-woven fabric carrier. The test strip can specifically detect organophosphorus and carbamate pesticides such as trichlorfon, malathion and phoxim through the enzyme activity inhibition principle, the detection limit of the pesticides such as phoxim is as low as 0.1 mg / kg, the developing intensity is in negative correlation with the pesticide concentration, and semi-quantitative judgment can be achieved. The preparation process is simple, the chitosan non-woven fabric is adopted as a fixed carrier, the material is environment-friendly and degradable, the enzyme activity recovery rate reaches 90%, the stability is excellent (the enzyme activity recovery rate can be preserved for 60 days at the temperature of 4 DEG C in a dark place), sample pretreatment is not needed in detection operation, a result can be obtained at the normal temperature for 15 min, the stability and repeatability are good, and the problems that a traditional instrument is high in detection cost and long in time consumption are solved; the method has a remarkable basic popularization value.

Description

Technical Field

[0001] This invention relates to the field of pesticide residue detection, specifically to a pesticide residue test strip based on chitosan nonwoven fabric, its preparation method, and its application. Background Technology

[0002] Organophosphates and carbamates are the two most commonly used classes of pesticides. They can inhibit the catalytic activity of acetylcholinesterase (AChE) in the human body through phosphorylation, preventing the breakdown of the neurotransmitter acetylcholine and leading to excessive accumulation of acetylcholine in the body, thus causing poisoning. Therefore, for the sake of consumers' health, pesticide residue testing of agricultural products, medicinal herbs, and food is a necessary measure.

[0003] Pesticide residue detection technologies are mainly divided into two categories: instrumental analysis methods and rapid detection methods. Instrumental analysis methods mainly include gas chromatography and high-performance liquid chromatography (HPLC). While instrumental analysis methods offer high accuracy, they suffer from significant drawbacks such as cumbersome experimental procedures, high costs, stringent operational requirements, strong dependence on testing sites and professionals, and long processing times. These limitations make them unsuitable for rapid screening needs in production sites and distribution channels, significantly restricting their application at the grassroots level. Rapid detection methods mainly include colorimetric methods, test strip methods, and biosensor methods. With their core advantages of convenience, efficiency, and low cost, rapid detection methods have become an important supplement to instrumental analysis methods. Rapid pesticide residue test strips developed based on enzyme inhibition methods enable on-site, real-time detection. They are easy to operate and have a short testing cycle, effectively compensating for the shortcomings of traditional detection technologies and providing a practical solution for rapid pesticide residue screening. These strips have significant value and promising application prospects for grassroots promotion.

[0004] The functional pad substrates of existing rapid pesticide residue test strips are mostly made of traditional materials such as nitrocellulose membranes and ordinary synthetic fibers. These substrates have inherent defects: First, the compatibility between enzyme molecules and the substrate is poor, resulting in low enzyme activity recovery rates and limiting detection sensitivity. Second, the substrate structure is not stable enough and is easily affected by environmental temperature and humidity, resulting in short shelf life and poor repeatability of the test strips. Third, the substrate has poor adsorption selectivity for impurities in the sample, which can easily cause color interference and affect the accuracy of result interpretation. Fourth, traditional synthetic substrates are difficult to degrade naturally, which is not environmentally friendly and contradicts the development trend of green detection. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a pesticide test strip based on chitosan nonwoven fabric, its preparation method, and its application.

[0006] The first objective of this invention is to provide a method for preparing a pesticide test strip, the method comprising the following steps: (1) Solution preparation. Enzyme solution: 50 mg acetylcholinesterase (AChE) dissolved in 100 mL of 0.05 mol / L PBS solution, concentration is 0.5 mg / mL; Substrate solution: 1 g thioacetylcholine iodide (ATCI) dissolved in 100 mL of distilled water, concentration is 10 mg / mL; Colorimetric solution: 0.4 g 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) dissolved in 100 mL of 0.05 mol / L PBS solution, concentration is 4 mg / mL. (2) Preparation of test strips. Cut 1 cm × 1 cm chitosan nonwoven fabric and 1 cm × 1 cm absorbent paper. Take 60 μL of enzyme solution and add 0.2 mol / L PBS to a total volume of 0.5 mL. Mix thoroughly to prepare an enzyme solution. Immerse one chitosan nonwoven fabric membrane in the enzyme solution and fix at 4℃ for 8 h. Remove the enzyme membrane and rinse several times with 0.2 mol / L PBS (pH=8.0) to obtain an immobilized enzyme pad. Store at 4℃ for later use. Immerse one chitosan nonwoven fabric membrane in the substrate solution and fix at 4℃ for 2 h. Remove the substrate to obtain an immobilized substrate pad. Store at 4℃ for later use. Immerse one chitosan nonwoven fabric membrane in the color developing solution. After the nonwoven fabric is completely wetted, remove the nonwoven fabric to obtain an immobilized color developing pad. Store at 4℃ for later use. The sample pad, barrier pad, and absorbent pad are all absorbent paper.

[0007] (3) Test strip assembly. The fixed reagent strips are laid flat on the PVA plate in the order of sample pad, enzyme pad, blocking pad, substrate pad, color development pad and adsorption pad by overlapping the edges and then fixed.

[0008] The second objective of this invention is to provide a pesticide residue test strip prepared using the above method.

[0009] The third objective of this invention is to provide a method for using the above-mentioned pesticide residue test strip. The detection steps include: adding 400 μL of sample processing solution to the sample pad and reacting it at 25°C for 15 min. The pesticide residue amount is determined by observing the intensity of the orange-yellow color of the color development pad. The color intensity is negatively correlated with the pesticide concentration. The color intensity of the negative control (without pesticide) is assigned a value of 100. A color intensity ≥80 is considered undetectable, a color intensity of 30-80 is considered low concentration residue (0.1-1.6 mg / kg), and a color intensity ≤30 is considered high concentration residue (≥1.6 mg / kg), thus achieving semi-quantitative determination.

[0010] Preferably, the samples include Chinese medicinal herbs and fruits and vegetables. The Chinese medicinal herbs are honeysuckle and wolfberry, and the fruits and vegetables are tomatoes, spinach, peppers, cucumbers, grapes, and apples. Among them, the fruit and vegetable samples do not require complicated pretreatment. They can be directly sampled or simply extracted with phosphate buffer, shaken for 2 minutes, and then allowed to stand to collect the supernatant for detection. The Chinese medicinal herb samples need to be extracted: honeysuckle samples are extracted with acetonitrile, impurities are removed, and the concentration is followed by redissolution with PBS solution for detection. Wolfberry samples are soaked in glacial acetic acid solution, extracted with acetonitrile, impurities are removed, and the concentration is followed by redissolution with PBS solution for detection.

[0011] The fourth objective of this invention is to provide the use of the above-mentioned pesticide residue test strip, which can be used for the rapid and accurate detection of organophosphate and carbamate pesticides.

[0012] The principle of enzyme inhibition is that organophosphates and carbamate pesticides can covalently bind to acetylcholinesterase (AChE) to form phosphorylated AChE that is difficult to hydrolyze, thereby inhibiting enzyme activity. Based on enzyme inhibition, we can develop test strips for rapid detection. Acetylcholinesterase (AChE) can induce the reaction of the substrate thioacetylcholine iodide to produce thiocholine. The sulfhydryl group on thiocholine can react with the chromogenic agent DTNB to produce a yellow product with a characteristic absorption peak at 412 nm. Therefore, by immobilizing AChE on a carrier medium and preparing a reaction substrate pad (containing thioacetylcholine iodide) and a detection zone (containing the chromogenic agent DTNB), the enzyme activity can be quantitatively characterized by detecting changes in absorbance, thus determining the degree of pesticide inhibition. Since there is a certain linear relationship between enzyme activity inhibition rate and pesticide concentration, quantitative detection of these two types of pesticide residues can be achieved.

[0013] Chitosan nonwoven fabric, as a natural polysaccharide-derived material, possesses excellent biocompatibility, structural stability, and environmental degradability. Its porous structure and surface-active groups enable efficient immobilization and activity retention of enzymes, substrates, and chromogenic agents, providing an ideal solution to many pain points of traditional substrates. Therefore, developing pesticide residue test strips using chitosan nonwoven fabric as the functional pad substrate, integrating high stability, high sensitivity, environmental friendliness, and convenience, can effectively compensate for the shortcomings of existing technologies, meet the practical needs of rapid on-site screening at the grassroots level, and possess significant technological innovation value and broad market prospects.

[0014] In use, the sample is dropped onto the sample pad. Due to the capillary action of the absorbent paper, the sample solution on the pad passes through the enzyme tablet. The acetylcholinesterase on the enzyme membrane quickly dissolves in the water and continues to move, passing through the blocking tablet and reaching the substrate tablet. There, it reacts with the substrate thioacetylcholine iodide to form thiocholine. Then, under the influence of water, it moves to the colorimetric pad and reacts with DTNB to form TNB, producing an orange-yellow indicator band. As the concentration of pesticide residue in the added sample solution increases, the color of the detection well gradually lightens from orange-yellow. The amount of pesticide residue in the sample can be determined based on the difference in color intensity.

[0015] The beneficial effects of the pesticide residue test strip provided in this application are as follows: Compared with the prior art, the test strip of this application has a simpler structure, a more regular shape, and is more convenient to operate than colorimetric cards on the market, resulting in more accurate detection results. This test strip, based on the principle of enzyme activity inhibition, can specifically detect organophosphorus and carbamate pesticides such as trichlorfon, malathion, and phoxim. The detection limit for pesticides such as phoxim is as low as 0.1 mg / kg, and the color intensity is negatively correlated with the pesticide concentration, enabling semi-quantitative determination. The preparation process of this invention is simple, using chitosan nonwoven fabric as a fixative carrier. The material is environmentally friendly and biodegradable, with an enzyme activity recovery rate of 90% and excellent stability (can be stored for 60 days at 4℃ in the dark). The detection operation requires no sample pretreatment, and results are available in 15 minutes at room temperature. It exhibits good stability and repeatability, solving the pain points of high cost and long time consumption of traditional instruments, and has significant value for grassroots promotion. Attached Figure Description

[0016] Figure 1 This is a diagram illustrating the construction of the pesticide test strip in Example 1; Figure 2 This is a schematic diagram illustrating the optimal material selection for immobilizing acetylcholinesterase in Example 2; Figure 3 This is a schematic diagram illustrating the selection of the amount of immobilized enzyme in Example 2; Figure 4 This is a diagram showing the optimization of acetylcholinesterase immobilization conditions in Example 2; Figure 5 This is a diagram showing the optimized reaction conditions for the immobilized enzyme in Example 2; Figure 6 This is a stability test diagram of the immobilized acetylcholinesterase in Example 2; Figure 7 This is a comparison graph showing the inhibitory effects of several pesticides on enzymes in Example 2; Figure 8 This is a schematic diagram illustrating the selection of materials, time, and concentration for immobilizing thioacetylcholine iodide in Example 2. Figure 9 This is a schematic diagram illustrating the material and time selection for fixing the DTNB in ​​Example 2; Figure 10 This is a graph from the accelerated experiment in Example 2; Figure 11 This is a graph showing the color development time of the test strip in Example 2; Figure 12 This is a schematic diagram illustrating the effect of the test strip in Example 2 on detecting three pesticides; Figure 13 Example 2: Paper strip detection of pesticide residues in Chinese medicinal materials; Figure 14 Example 2: Test strips were used to detect pesticide residues in vegetables after pesticide spraying. Specific implementation methods

[0017] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and are not intended to limit the scope of this application.

[0018] The reagents and equipment used in the following examples can all be purchased through commercial channels.

[0019] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0020] Example 1: Preparation of test strips A test strip for rapid and accurate detection of organophosphates and carbamates was prepared. The preparation method includes the following steps: (1) Solution preparation. Enzyme solution: 50 mg acetylcholinesterase (AChE) dissolved in 100 mL of 0.05 mol / L PBS solution, concentration is 0.5 mg / mL; Substrate solution: 1 g thioacetylcholine iodide (ATCI) dissolved in 100 mL of distilled water, concentration is 10 mg / mL; Colorimetric solution: 0.4 g 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) dissolved in 100 mL of 0.05 mol / L PBS solution, concentration is 4 mg / mL. (2) Preparation of test strips. Cut 1 cm × 1 cm chitosan nonwoven fabric and 1 cm × 1 cm absorbent paper. Take 60 μL of enzyme solution and add 0.2 mol / L PBS to a total volume of 0.5 mL. Mix thoroughly to prepare an enzyme solution. Immerse one chitosan nonwoven fabric membrane in the enzyme solution and fix at 4℃ for 8 h. Remove the enzyme membrane and rinse several times with 0.2 mol / L PBS (pH=8.0) to obtain an immobilized enzyme pad. Store at 4℃ for later use. Immerse one chitosan nonwoven fabric membrane in the substrate solution and fix at 4℃ for 2 h. Remove the substrate to obtain an immobilized substrate pad. Store at 4℃ for later use. Immerse one chitosan nonwoven fabric membrane in the color developing solution. After the nonwoven fabric is completely wetted, remove the nonwoven fabric to obtain an immobilized color developing pad. Store at 4℃ for later use. The sample pad, barrier pad, and absorbent pad are all absorbent paper.

[0021] (3) Test strip assembly. For example... Figure 1 As shown, the fixed reagent pads are laid flat on the PVA plate in the order of sample pad, enzyme pad, blocking pad, substrate pad, color development pad, and adsorption pad by overlapping the edges. Example

[0022] 1 Experimental Methods 1.1 Enzyme activity assay With a slight modification to the Ellman method, free enzyme activity was determined as follows: 60 μL of AChE solution and 3.64 mL of 0.2 mol / L, pH 8.0 PBS were added and incubated at 37°C for 3 min. Then, 100 μL of DTNB solution and 200 μL of ATCI solution were added, and the mixture was incubated at 37°C for 3 min. The reaction was terminated by adding an equal volume of anhydrous ethanol. The absorbance was measured at 412 nm. One unit of enzyme activity was defined as a change of 0.01 μm in absorbance at 412 nm under the conditions of 37°C, 0.2 mol / L, pH 8.0 PBS for 3 min. A blank control was performed using distilled water.

[0023] Replace the acetylcholinesterase solution with immobilized enzyme tablets and measure the immobilized enzyme activity.

[0024] 1.2 Calculation of enzyme activity recovery rate The enzyme activity recovery rate (R) is calculated using the formula R = ΔA1 / ΔA2 × 100%, where ΔA1 refers to the total activity of the immobilized enzyme and ΔA2 refers to the total activity of the added enzyme. 1.3 Calculation of enzyme activity inhibition rate Inhibition rate (%) = [(OD control - OD sample) / OD control] × 100, where OD control refers to the absorbance without pesticides and OD sample refers to the absorbance with pesticides.

[0025] 1.4 Quantification of Color Rendering Intensity Color intensity was quantified by acquiring digital images and analyzing them using Adobe Illustrator software. The negative control (orange-yellow without any inhibitors) was assigned a value of 100, and the positive control (pale yellow without enzymes) was assigned a value of 0. The experimental design was completely randomized, with each experiment repeated three times.

[0026] 1.5 Application of test strips in detecting pesticide residues in Chinese medicinal herbs and fruit and vegetable samples Honeysuckle powder (passed through a No. 3 sieve), accurately weighed, placed in a 50 mL stoppered centrifuge tube, add 5 mL of water, shake well, let stand for 30 min, add 2.5 g of sodium chloride, immediately shake to disperse, then add 20 mL of acetonitrile, homogenize for 2 min (12000 r / min), centrifuge (4000 r / min) for 5 min, separate the supernatant, add 25 mL of acetonitrile to the precipitate, homogenize for 1 min, centrifuge (4000 r / min) for 5 min, combine the supernatants from the two extractions, dilute with acetonitrile to 50 mL, shake well, add 0.5 g of diatomaceous earth, 0.1 g of C18, 0.05 g of activated carbon, and 0.03 g of sodium chloride to every 5 mL of liquid, homogenize for 2 min (speed not less than 12000 r / min), centrifuge (4000 r / min) for 5 min, remove impurities until clear, if there is still some yellow, add a little diatomaceous earth, activated carbon, and C18, take 10 Concentrate the supernatant to 0.5 mL, then add 9.5 mL of PBS solution (containing 0.05% Tween-20, 1% BSA, and pesticide detection-specific PBS powder) to 10 mL.

[0027] Goji berry: Accurately weigh 3 g of the test sample powder (passed through a No. 3 sieve; the goji berry sample must be frozen and rapidly pulverized), place it in a 50 mL stoppered centrifuge tube, add 15 mL of 1% glacial acetic acid solution, vortex to disperse evenly, let stand for 30 min, accurately add 15 mL of acetonitrile, vortex to mix, place on a shaker and shake vigorously (500 r / min) for 5 min, place in an ice bath for 20 min, add 7.5 g of a mixture of anhydrous magnesium sulfate and anhydrous sodium acetate powder (4:1), shake immediately to disperse, then place on a shaker and shake vigorously for 3 min, centrifuge (4000 r / min) for 5 min, accurately pipette 5 mL of the supernatant, dilute with acetonitrile to 20 mL, shake well, add 100 mg of diatomaceous earth, 50 mg of C18, and ≤10 mg of activated carbon per 1 mL of liquid, homogenize for 2 min (speed not less than 12000 r / min), centrifuge (4000 r / min) for 5 min. Min, remove impurities until clear. If there is still some yellow, add a little diatomaceous earth, activated carbon, and C18. Take 10 mL of supernatant and concentrate to 0.5 mL. Add 9.5 mL of PBS solution (containing 0.05% Tween-20, 1% BSA, and pesticide detection-specific PBS powder) to 10 mL.

[0028] Select frequently consumed fruits and vegetables, gently wipe off surface soil, and spray with different concentrations of phoxim pesticide at a rate of 1 g / mL. The phoxim solution concentrations are 0, 2, 4, 8, 16, and 20 mg / kg. After standing at room temperature for 24 hours, cut into approximately 1 cm pieces, weigh 2 g of sample, place in a beaker, add 5 mL of phosphate buffer, shake for 2 min, and let stand. The supernatant is reserved for analysis. For analysis, one sample slide has 400 μL of PBS solution added as a negative control, and the other samples are added sequentially with the fruit and vegetable sample solution. The reaction is carried out at room temperature for 15 min.

[0029] 2. Statistical Analysis The significance of differences was analyzed using SPSS software (Duncan method and LSD method) and Graph Pad (Fisher's LSD method). Graph Pad was used to create graphs and tables. Note: Letter notation: a, b, c, d, etc., are used to indicate significance. Asterisk notation: one asterisk (*) indicates a significance level of 0.05, two asterisks (**) indicate a significance level of 0.01, and three asterisks (***) indicate a significance level of 0.001.

[0030] 3 Results Analysis 3.1 Optimal material for immobilizing acetylcholinesterase The optimal immobilization material for enzymes should be bioinert, so that it is not affected by bioactive substances and can achieve complete adsorption and release. Therefore, Figure 2 A composite membrane (Fhm) composed of glass fiber (RB65), polyester fiber (VL78), absorbent paper (SX18), chitosan nonwoven fabric (Kjt), and 50% Tencel fiber and 50% chitosan was used as the immobilization material. The recovery rate of enzyme activity of enzyme tablets made of different materials was measured. The results showed that the enzyme activity recovery rate varied depending on the material used as the enzyme tablet. The enzyme activity recovery rate of chitosan nonwoven fabric was the highest, reaching 90%. Figure 2 B). Enzyme activity recovery was measured after incubation for different times, and the results showed that it had little effect on enzyme activity. Therefore, chitosan nonwoven fabric (Kjt) was determined to be the optimal enzyme immobilization material.

[0031] 3.2 Immobilized enzyme dosage Figure 3 The effect of acetylcholinesterase (AChE) dosage on immobilization efficiency is shown. Insufficient enzyme dosage results in insufficient enzyme adsorption on the membrane surface. As the enzyme dosage increases, the amount of enzyme adsorbed on the membrane surface gradually increases, manifested as a gradual increase in the enzyme activity of the immobilized enzyme. At an enzyme dosage of 60 μL, the membrane surface reaches adsorption saturation; further increases in enzyme dosage do not increase the enzyme activity of the immobilized enzyme membrane.

[0032] 3.3 Optimization of acetylcholinesterase immobilization conditions Different pH values ​​affect the charged state of chitosan nonwoven fabrics, thus influencing enzyme immobilization efficiency. From Figure 4 As can be seen from A, when the pH value is 5.0-8.0, the activity of the immobilized enzyme gradually increases with the increase of pH value. When the pH value is higher than 8.0, the enzyme activity drops sharply, which may be because the excessively high pH value inactivates the enzyme.

[0033] Immobilization time has a direct impact on the immobilization effect. From Figure 4 B shows that immobilizing the enzyme solution at 4℃ for 2-4 hours is a short time, which prevents the enzyme molecules from fully adsorbing and binding to the membrane, resulting in a smaller amount of immobilized enzyme and lower immobilized enzyme activity. As the immobilization time increases, the activity of the immobilized enzyme increases, reaching its highest level after 8 hours of immobilization. When the immobilization time is extended to 24-48 hours, the enzyme activity decreases slightly, which is due to the enzyme denaturing and becoming inactive after prolonged exposure to air.

[0034] Acetylcholinesterase is sensitive to temperature. When the immobilization temperature is too high, the enzyme is easily inactivated, and the immobilization effect is not ideal. When the temperature is too low, the conditions are difficult to control. Figure 4 C indicates that the enzyme activity is highest at 4℃, and gradually decreases with increasing temperature. At 40℃, the enzyme has almost no activity. Therefore, the immobilization temperature of acetylcholinesterase should be 4℃.

[0035] 3.4 Optimization of reaction conditions for immobilized enzymes The pH of the reaction modulates the binding affinity of the enzyme to its substrate by altering the dissociation state of amino acid residues at the enzyme's active site. Figure 5 As shown in Figure A, enzyme activity is lower at lower pH levels, indicating that an acidic environment inhibits enzyme activity and is unfavorable for enzymatic reactions. Enzyme activity gradually increases with increasing pH, reaching its maximum at pH 8.0. Beyond pH 8.0, enzyme activity decreases with further increases in pH. The optimal pH for the free enzyme is 8.0, which is not significantly different from that of the immobilized enzyme. This indicates that immobilization did not alter the dissociation groups of AChE.

[0036] Reaction temperature affects the rate of molecular thermal motion, thus balancing the conformational stability and catalytic kinetic efficiency of the enzyme. Following enzyme activity assay methods, reactions were conducted at 20℃, 30℃, 37℃, and 45℃ to determine the effect of temperature on the activity of the immobilized enzyme. The results are as follows: Figure 5 As shown in B, the optimal reaction temperature for immobilized enzymes is 37℃.

[0037] 3.5 Stability test of immobilized acetylcholinesterase The activity of the immobilized enzyme was continuously measured according to the enzyme activity assay method. Figure 6 A) The enzyme activity was high in the first 4 measurements; after the 5th measurement, the enzyme activity gradually decreased, indicating that the immobilized enzyme tablets can be used 4-5 times in the absence of inhibitors.

[0038] The enzymes were stored at 4°C and room temperature (25°C) for one month, and the activities of immobilized and free enzymes were measured every few days. A graph was plotted with relative activity on the ordinate and time on the x-axis. Figure 6 As shown in B, the immobilized enzyme exhibits excellent stability at both 4°C and room temperature, with relative activities of 82% and 63% respectively after one month of storage. In contrast, the free enzyme shows relatively poor stability, with rapid activity loss at room temperature and a 50% activity loss after 18 days at 4°C. Therefore, the optimal storage temperature is 4°C.

[0039] 3.6 Inhibitory effects of several pesticides on enzyme tablets Different concentrations of trichlorfon, malathion, and phoxim were prepared to conduct inhibition tests on free and immobilized enzymes. Figure 7 Plot the inhibition rate on the ordinate and the concentration on the abscissa. The comparison of the inhibition of the two enzymes by the three pesticides shows that the immobilized enzyme is much more sensitive to pesticides than the free enzyme, with a higher inhibition rate at the same concentration.

[0040] 3.7 Selection of materials, time, and concentration for immobilizing thioacetylcholine iodide For the substrate material, the recovered substrate was reacted with the enzyme, and the absorbance was measured. From Figure 8As can be seen from A, the chitosan nonwoven fabric has the highest absorbance value at 412 nm, indicating that the chitosan nonwoven fabric is the most suitable substrate fixation material.

[0041] from Figure 8 As can be seen from B, the enzyme activity varies with different fixation times, with the optimal fixation time being 2 hours.

[0042] The results of substrate concentration optimization are as follows: Figure 8 As shown in Figure C, the color intensity tends to increase with increasing substrate concentration. Therefore, the optimal substrate concentration was determined to be the stock solution concentration (10 mg / mL).

[0043] 3.8 Selection of materials and time for fixing the color developer DTNB Figure 9 The conclusion drawn by A is that chitosan nonwoven fabric (Kjt) has a better retention capacity than nitrocellulose membrane (NC membrane) when used as a DTNB immobilization carrier. This indicates that the material properties of Kjt are more suitable for the immobilization requirements of DTNB and can better maintain the bioactivity of DTNB, making it a more promising immobilization material.

[0044] from Figure 9 As can be seen from B, in the concentration range of 0.5-4 mg / mL, the DTNB concentration is positively correlated with the color intensity of the system. After the concentration exceeds 4 mg / mL, the color intensity reaches saturation and no longer changes significantly with increasing concentration, reflecting that the substrate concentration is the critical threshold of color response in this detection system.

[0045] 3.9 Accelerated Test Strip Experiment Temperature and light are the core environmental factors affecting the activity stability of test strips, and their synergistic effect significantly regulates the activity decay rate. Figure 10 The bar charts ABCD represent the color intensity quantification (ABCD represent the activity of the test strips under 37℃ light, 25℃ light, 25℃ in the dark, and 4℃ in the dark, respectively), while EFGH represent the accelerated color development results of the test strips under different temperature and light conditions. The bar charts and color development results complement each other, revealing the experimental results comprehensively from different dimensions.

[0046] Depend on Figure 10 The enzyme activities of ABCD and Figure 10The color of the EFGH test strips indicates that the activity of the test strips decayed fastest under light exposure at 37℃, with the color intensity dropping to an extremely low level within 5 days. At 25℃, the activity retention of the light-protected group (maintaining a certain level of activity for 28 days) was significantly better than that of the light-exposed group (activity decreased sharply after 14 days). The test strips exhibited the best stability at 4℃ in the dark, retaining relatively high activity even after 60 days. Accelerated experiments confirmed that the activity decay rate after 2.5 days of light exposure at 37℃ was similar to that after 28 days of light protection at 25℃ and 60 days of light protection at 4℃, and the decay effect after 1 day of light exposure was approximately equivalent to 9.3 days of light protection. These results indicate that low temperature and light protection can significantly delay decay, making it the optimal storage condition.

[0047] 3.10 Determination of color development time The test strips were placed at 4℃, 25℃, and 37℃ for reaction, and the real-time detection results were as follows: Figure 11 As shown, regardless of whether the reaction was conducted at 4℃, 25℃, or 37℃, the test strip reached its maximum color intensity within 15 minutes. At 4℃, the color intensity response was weak; at 25℃ and 37℃, the difference in color intensity was not significant; and at 37℃, the test strip evaporated rapidly, requiring more liquid solution for the reaction. Therefore, the optimal reaction time was determined to be 15 minutes, and the optimal operating temperature was determined to be 25℃.

[0048] 3.11 Test strips were used to detect the effects of three pesticides. Figure 12 The test results showed that the self-made test strip exhibited concentration-dependent detection response capabilities for three pesticides: trichlorfon, malathion, and phoxim. Overall, a negative correlation was observed between pesticide concentration and the color intensity of the test strip: as the pesticide concentration increased from 0.1 mg / kg to 20 mg / kg, the color of the test strip gradually lightened, and at the high concentration of 20 mg / kg, the color intensities corresponding to the three pesticides tended to be consistent, indicating that the test strip can achieve semi-quantitative concentration gradient discrimination for these three pesticides. Meanwhile, there were significant differences in detection sensitivity among the different pesticides. The test strip was more sensitive to phoxim, showing a significant decrease in color intensity in the low concentration range of 0.1-0.2 mg / kg, while the color decay of trichlorfon and malathion was relatively gradual, reflecting that the test strip's detection sensitivity for phoxim was superior to that of trichlorfon and malathion.

[0049] 3.12 Paper strip detection of pesticide residues in Chinese medicinal herbs Figure 13The results of colorimetric analysis of honeysuckle and wolfberry samples using a self-made test strip and a commercially available test card are presented. In terms of colorimetric characteristics, the self-made test strip produced a pure yellow hue for both types of medicinal herbs, with a uniform color and no interference from other colors, clearly reflecting the sample's detection response characteristics. In contrast, the commercially available blue test card showed significant interference in the colorimetric area due to impurities; the blue hue was mixed with non-target colorimetric substances, significantly reducing the recognizability and intuitiveness of the colorimetric results. Comparing the colorimetric performance of the two types of materials, the self-made test strip has a significant advantage in colorimetric purity, effectively avoiding interference from impurities. Both the self-made test strip and the commercially available test card produced detection responses for honeysuckle and wolfberry samples, with good repeatability.

[0050] 3.13 Test strips for detecting pesticide residues in vegetables after pesticide spraying Figure 14 A self-made test strip and a commercially available colorimetric card were used to detect phoxim in six kinds of fruits and vegetables. The color development matched the (-)→(+)→(++) gradient of the colorimetric card. The results showed that both detection tools exhibited concentration-dependent responses to phoxim, and the detection sensitivity varied among different fruits and vegetables. Both tools can achieve semi-quantitative standardized determination of phoxim content and are suitable for rapid screening of phoxim residues in various fruits and vegetables. The self-made test strip showed higher sensitivity than the commercially available card, with clearer color change levels, while the gradient response of the commercially available card was more ambiguous.

[0051] The test strips of this application can be used to detect various organophosphorus pesticides and carbamate pesticides.

[0052] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing a pesticide residue detection test strip, characterized in that... The method includes the following steps: (1) Solution preparation: Enzyme solution: 50 mg acetylcholinesterase was dissolved in 100 mL of 0.05 mol / L PBS solution, with a concentration of 0.5 mg / mL; Substrate solution: 1 g thioacetylcholine iodide was dissolved in 100 mL of distilled water, with a concentration of 10 mg / mL; Colorimetric solution: 0.4 g 5,5'-dithiobis(2-nitrobenzoic acid) was dissolved in 100 mL of 0.05 mol / L PBS solution, with a concentration of 4 mg / mL; (2) Preparation of test strips: Cut 1 cm × 1 cm chitosan nonwoven fabric and absorbent paper. Take 60 μL of enzyme solution and add 0.2 mol / L PBS to a total volume of 0.5 mL. Mix well to prepare enzyme solution. Take one chitosan nonwoven fabric membrane and immerse it in the enzyme solution. Fix it at 4℃ for 8 h. Take out the enzyme membrane and rinse it several times with 0.2 mol / L PBS, pH=8.0 to obtain immobilized enzyme pad. Store it at 4℃ for later use. Take one chitosan nonwoven fabric membrane and immerse it in the substrate solution. Fix it at 4℃ for 2 h. Take out the substrate to obtain immobilized substrate pad. Store it at 4℃ for later use. Take one chitosan nonwoven fabric membrane and immerse it in the color developing solution. After the nonwoven fabric is completely wetted, take out the nonwoven fabric to obtain immobilized color developing pad. Store it at 4℃ for later use. The sample pad, barrier pad and absorbent pad are all absorbent paper. (3) Test strip assembly: The fixed reagent strips are fixed onto the PVA plate in the order of sample pad, enzyme pad, blocking pad, substrate pad, color development pad and adsorption pad by overlapping the edges.

2. A pesticide residue detection test strip, characterized in that... The test strip is prepared using the method described in claim 1.

3. The pesticide residue detection test strip according to claim 2, characterized in that... The test strips are used for the semi-quantitative detection of organophosphorus and carbamate pesticides.

4. A method of using a pesticide residue detection test strip as described in claim 2, characterized in that... The method is as follows: 400 μL of sample treatment solution is added to the sample pad and reacted at 25℃ for 15 min. The pesticide residue is determined by observing the intensity of the orange-yellow color of the color development pad. The color intensity is negatively correlated with the pesticide concentration. The color intensity of the negative control is assigned a value of 100. A color intensity ≥80 is considered undetectable. A color intensity of 30-80 indicates a residue concentration of 0.1-1.6 mg / kg, which is considered a low concentration residue. A color intensity ≤30 indicates a residue concentration ≥1.6 mg / kg, which is considered a high concentration residue.

5. The method of using the pesticide residue detection test strip according to claim 4, characterized in that... The samples consisted of Chinese medicinal herbs and fruits and vegetables.

6. The method of using the pesticide residue detection test strip according to claim 5, characterized in that... The method for obtaining the fruit and vegetable sample processing solution is to directly sample or extract with phosphate buffer, shake for 2 minutes, and then let it stand to collect the supernatant.

7. The method of using the pesticide residue detection test strip according to claim 5, characterized in that... The Chinese herbal sample is honeysuckle, and the sample processing solution is obtained by extracting honeysuckle with acetonitrile, removing impurities, concentrating it, and then reconstituted it with PBS solution.

8. The method of using the pesticide residue detection test strip according to claim 5, characterized in that... The Chinese herbal sample was wolfberry, and the sample processing solution was obtained by soaking wolfberry in glacial acetic acid solution, extracting with acetonitrile, removing impurities, concentrating, and then redissolving with PBS solution.

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